The overall goal of this project is to elucidate the changes that occur within T helper (Th) cells when suppressed by regulatory T cells (Tregs) and identify the molecular mechanisms underlying Treg mediated inactivation of effector T cell functions. The specific aims of this project include (1) characterizing the role of Nuclear Factor of Activated T cells (NFAT) within suppressed T cells, identifying the molecular mechanisms of activation, transcriptional partners and the interaction with downstream targets;and (2) determining if Treg function is controlled by silencing of the interleukin (IL-)2 gene. To approach the first aim, immunofluorescence will reveal if NFAT is retained in the cytoplasm or translocated across the membrane, where it is active and can induce the expression of proteins known to suppress T cell activation. Electrophoretic mobility shift assays will be used to determine if active NFAT proteins can bind DNA in the nucleus of suppressed Th cells, in the absence of AP-1. Subsequently, chromatin immunoprecipitation (ChIP) will be performed on suppressed Th1 cells to determine if activation by NFAT involves direct recruitment of this protein to the promoter regions of T cell inactivating genes. By completing our goals in aim one we intend to uncover the transcriptional regulation of NFAT activation within suppressed T cells and elucidate gene expression patterns that are necessary for the induction and maintenance of the unresponsive state. For the second aim, chromatin immunoprecipitation assays will determine if epigenetic modifications regulate IL-2expression in stimulated Tregs and suppressed Th cells. To study the role of histone deacetylation in the suppressor activity and function of Tregs, isolated Tregs from BALB/c mice will be cocultured with TCR transgenic D011.10 mice and stimulated in the presence or absence of the histone deacetylase inhibitor, Trichostatin-A. Our overall goal in aim two is to determine the role that chromatin remodeling plays in the induction of T cell tolerance and investigate the mechanisms that regulate cytokine expression in Tregs and in Treg-mediated suppression of Th cells. A clear understanding of how theIL-2 locus is regulated in both Tregs and suppressed cells will prove invaluable in elucidating Treg behavior and function. By uncovering the mechanisms of Treg-mediated suppression of effector T cell function, novel therapies inducing immune tolerance can be developed against a host of autoimmune diseases and cancers. This project is relevant to the public at large because of the importance this cell (Treg) plays in maintaining a healthy immune system. By studying the behavior of this cell type (Treg), the causes of different human diseases can be better understood and new therapies developed.